Protein kinase C, PKC, belongs to a family of widely distributed signal transduction proteins important for cell growth, differentiation, and other responses. PKC is activated by growth factors, hormones, and other external messengers via stimulation of phospholipase C and is responsible for the generation of the second messengers, inositol triphosphate and diacylglycerol. All members of the PKC family share significant sequence homology and perform signal transduction via protein phosphorylation. Almost all cell types express one or more isoforms of PKC.
The activity of an endogenous inhibitor of PKC has been detected in bovine, avian, murine, and human tissues. The first complete primary structure of an endogenous inhibitor of protein kinase C, PKCI-1, was derived from bovine brain. (Pearson, J. D. et al. (1990) J. Biol. Chem. 265: 4583-4591.) PKCI-1 has a specific site of interaction with PKC and inhibits the ability of PKC to carry out phosphorylation. In addition to the bovine sequence, a complete amino acid sequence of PKCI-1 has been deduced from the maize, rat, and human genes. (Simpson G. G. et al. (1994) Biochim. Biophys. Acta. 1222: 306-308; Waller S. J. and Murphy D (1994) GenBank Accession number GI 493051; and Brzoska P. M. et al. (1995) Proc. Natl. Acad. Sci. 92: 7824-7828.) The bovine PKCI-1 was shown to be a zinc (Zn) binding protein. (Simpson et al., supra.) The site of Zn binding has been localized to an 11 amino acid fragment. (Mozier et al. (1991) FEBS Lett. 279:14-18.) The Zn binding domain is conserved among other PKCI-1 molecules.
PKC activation plays a significant role in multidrug resistance (MDR), a major contributing factor in the failure of many chemotherapeutic cancer regimens. (Grunicke H. et al. (1994) Ann. Hematol. 69:S1-S6.) PKC regulates proteins, such as fos, jun, glutathione S-transferase, deoxy-thymidine monophosphate synthase, metallothionein, and mdr-1-encoded P-glycoprotein, that act to make cancer cells drug resistant. This regulation works through phosphorylation and results in increased transcription of the genes encoding these proteins. Conversely, PKC inhibitors or antagonists interfere with the phosphorylation function of PKC which reduces the expression of genes that mediate MDR. In addition to its role in MDR, PKC activation is often a critical event in tumor promotion. (O'Brian C. A. and Ward N. E. (1989) Cancer Metastasis Rev. 8: 199-214; O'Brian C. A. et al. (1995) Prog. Clin. Biol. Res. 391: 117-120.) For example, PKC alpha phosphorylates and activates the growth promoting gene Raf-1. (Kolch W. et al. (1993) Nature 364: 249-252.) Without PKC induced Raf-1 activation, the ability to transform NIH3T3 cells is greatly inhibited. Thus, PKC inhibitors (PKCI) are promising agents for cancer treatment.
PKC participates in the sequence of molecular events that underlie learning and memory. (Olds J. L. and Alkon D. L. (1991) New Biol. 3: 27-35.) The cellular distribution of PKCs changes as a result of memory storage in cells that have been demonstrated to act in memory and learning. (Saito N. et al. (1994) Brain Res. 656: 245-256.) PKC gamma mutant mice exhibit mild deficits in spatial and contextual learning, further implicating PKCs in learning and memory. (Abeliovich A. et al. (1993) Cell 75: 1263-1271.) Memory deterioration is one of the main characteristics and earliest signs of Alzheimer's disease.
PKCs can act in several ways to stimulate programmed cell death, also known as apoptosis, in various cell types. PKCs can block the activation of other calcium-dependent enzymes triggering apoptosis. (Lucas M. et al. (1995) Gen. Pharmacol. 26: 881-887.) Activation of phosphatases by ceramide and inhibition of PKC by sphingosine mediates the sphingomyelin pathway to apoptosis. A putative PKC target, p34cdc2, can act to stimulate apoptosis when its activity is uncoupled from the completion of DNA replication. In addition, p21Ras mediates proliferative responses and also renders cells susceptible to apoptosis after inhibition of PKC activity. (Chen C. Y. et al. (1996) J. Biol. Chem. 271: 2376-2379.) T lymphocytes of mice in which the Fas mediated apoptosis pathway has been knocked out rely on a PKC dependent apoptotic mechanism for selection against self-reactive T-cells. (Ohkusu K. et al. (1995) Eur. J. Immunol. 25: 3180-3186.) Thus, PKC has a role in immune cell development. Saikosponin b2 induces apoptosis in B16 melanoma cells by down regulation of PKC activity. (Zong et al. (1996) Biochem. Biophys. Res. Commun. 219: 480-485.) Furthermore, proteolytic activation of PKC delta by an ICE-like protease timulates apoptosis in human tumor cell line U-937. (Emoto Y. et al. (1995) EMBO J. 14: 6148-5156.)
There is significant evidence suggesting that PKC inhibitors may modify PKC activity in normal or diseased cells. For example, PKC inhibitors have been shown to inhibit the growth of cancer cells both in vitro and in vivo. (Levitzki A. (1994) Eur. J. Biochem. 226: 1-13.) The synthetic PKC inhibitor bisindolylmaleimide GF109203X (bisi) can affect the neuroblastoma cell line Neuro-2A in one of two ways. Without serum, neurite outgrowth is potentiated by bisi, while with serum, apoptosis occurs. (Behrens M. M. et al. Cell Growth Differ. (1995) 6: 1375-1380.) Other PKC inhibitors including hypericin, staurosporine, tamoxifen, and the phorbol ester PMA induce apoptosis in the human neuroblastoma cell line SK-N-SH. (Zhang W. et al. (1995) Cancer Lett. 96: 31-35.) PKC inhibitors may have utility in immune disorders as well. The PKC inhibitor H-7 induces apoptosis in Fas.sup.- mouse T lymphocytes. (Ohkusu et al. supra.)
The discovery of a new human protein kinase C inhibitor and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cancer, autoimmune disorders, and cognitive disorders.